The growing emergency of multi-drug resistant-bacteria is a global concern, mostly in those countries where antibiotics are widely used in clinics. A number of pathogens like Staphylococcus aureus, Staphylococcus epidermidis, Mycobacterium tuberculosis, some enterococci, Pseudomonas aeruginosa and many other bacteria have developed resistance against most traditional antibiotics as well as against those of new generation (Wenzel and Edmond 2000). It has therefore become increasingly important to develop new antibiotics. This demand urges the community of researchers and the pharmaceutical companies to consider new antimicrobial agents. Antimicrobial peptides are considered one of the best alternative to traditional antibiotics which generally cause the selection of resistant bacteria (Hancock and Sahl, 2006). Most antibacterial peptides are components of the innate immunity of animals, including humans, plants and fungi (Zasloff, 2002). They usually consist of 6-50 amino acid residues and have a positive net charge. Cationic peptides interact selectively with anionic bacterial membranes and with other negatively charged structures such as LPS and DNA. Eukaryotic membranes, in their external layer, are normally less negatively charged than bacteria's, and, differently from bacterial membrane, they are also stabilized by cholesterol molecules. These differences are the basis of cationic peptides' specificity. The mechanism of action of cationic antimicrobial peptides is consequently due to their specific binding to bacterial membranes, which provokes cell permeation and, in some cases, metabolic pathways inhibition.
Many studies then, aimed to the identification and characterization of antimicrobial peptide sequences by studying their mechanism of action, their toxicity for eukaryotic cells and their therapeutic efficacy when administered topically or systemically. Unfortunately, two main problems hindered the development of antimicrobial peptide drugs so far. The first is that selectivity of natural antimicrobial peptides for bacteria is generally too low and they appear to be very toxic for eukaryotic cells, particularly erythrocytes, generating a high level of haemolysis. The second is linked to the generally short half-life of peptides in vivo. These are the main reasons for which only few cationic peptides reached the market in the last 10 years (polymyxin and daptomycin are two successful examples).
A few years ago, researchers began to concentrate on the identification of novel peptide sequences of non-natural origin, selected in the laboratory by rational design or screening of combinatorial libraries. The aim was to find peptides with better biological properties in terms of general toxicity and specificity for bacteria and improved half-life for drug development.
In the inventors' laboratory, non-natural peptide sequences were identified, which showed a strong antimicrobial activity especially against Gram-negative bacteria (Pini et al, 2005; Pini et al., 2007; Pini et al., 2010). The last improved version of these peptides was called M33 (sequence KKIRVIRLSA, SEQ ID NO: 1; WO 2010/038220 A1) and it was tested in vitro for its capability to neutralize bacterial LPS and in vivo for its antibacterial and anti-inflammatory activity in sepsis animal models (Pini et al., 2010). Peptide M33 appeared selective for Gram-negative bacteria with a slight activity against only two strains of the Gram-positive Staphylococcus aureus. The peptide M33 was used and described in Pini et al., 2010 and in WO2010/038220A1 always with amino acids in L configuration (L-M33). Surprisingly, the synthesis of the same peptide sequence wide all the amino acids in D-configuration (D-M33) produced not only a better activity for some Gram-negative bacteria but also a strong activity against a panel of Gram-positive bacteria and fungi for which the L-M33 was not active at all. This result is completely unexpected, in that substitution of L-amino acids with their D configuration is known to increase peptide stability but the effect on peptide selectivity was not predictable.